Protease inhibitors may be classified either by the type of protease they inhibit, or by their mechanism of action. In 2004 Rawlings and colleagues introduced a classification of protease inhibitors based on similarities detectable at the level of amino acid sequence. This classification initially identified 48 families of inhibitors that could be grouped into 26 related superfamily (or clans) by their structure. According to the MEROPS database there are now 81 families of inhibitors. These families are named with an I followed by a number, for example, I14 contains hirudin-like inhibitors.
Proteinase propeptide inhibitors (sometimes referred to as activation peptides) are responsible for the modulation of folding and activity of the peptidase pro-enzyme or zymogen. The pro-segment docks into the enzyme, shielding the substrate binding site, thereby promoting inhibition of the enzyme. Several such propeptides share a similar topology, despite often low sequence identities. The propeptide region has an open-sandwich antiparallel-alpha/antiparallel-beta fold, with two alpha-helices and four beta-strands with a (beta/alpha/beta)x2 topology.
The peptidase inhibitor I9 family contains the propeptide domain at the N-terminus of peptidases belonging to MEROPS family S8A, subtilisins. The propeptide is removed by proteolytic cleavage; removal activating the enzyme.
The inhibitor I29 domain, which belongs to MEROPS peptidase inhibitor family I29, is found at the N-terminus of a variety of peptidase precursors that belong to MEROPS peptidase subfamily C1A; these include cathepsin L, papain, and procaricain. It forms an alpha-helical domain that runs through the substrate-binding site, preventing access. Removal of this region by proteolytic cleavage results in activation of the enzyme. This domain is also found, in one or more copies, in a variety of cysteine peptidase inhibitors such as salarin.
The structure of SMPI has been determined. It has 102 amino acid residues with two disulphide bridges and specifically inhibits metalloproteinases such as thermolysin, which belongs to MEROPS peptidase family M4. SMPI is composed of two beta-sheets, each consisting of four antiparallel beta-strands. The structure can be considered as two Greek key motifs with 2-fold internal symmetry, a Greek key beta-barrel. One unique structural feature found in SMPI is in its extension between the first and second strands of the second Greek key motif which is known to be involved in the inhibitory activity of SMPI. In the absence of sequence similarity, the SMPI structure shows clear similarity to both domains of the eye lens crystallins, both domains of the calcium sensor protein-S, as well as the single-domain yeast killer toxin. The yeast killer toxin structure was thought to be a precursor of the two-domain beta gamma-crystallin proteins, because of its structural similarity to each domain of the beta gamma-crystallins. SMPI thus provides another example of a single-domain protein structure that corresponds to the ancestral fold from which the two-domain proteins in the beta gamma-crystallin superfamily are believed to have evolved.
Inhibitor family I48 includes clitocypin, which binds and inhibits cysteine proteinases. It has no similarity to any other known cysteine proteinase inhibitors but bears some similarity to a lectin-like family of proteins from mushrooms.
^Jain SC, Shinde U, Li Y, Inouye M, Berman HM (November 1998). "The crystal structure of an autoprocessed Ser221Cys-subtilisin E-propeptide complex at 2.0 A resolution". J. Mol. Biol. 284 (1): 137–44. doi:10.1006/jmbi.1998.2161. PMID9811547.
^Groves MR, Taylor MA, Scott M, Cummings NJ, Pickersgill RW, Jenkins JA (October 1996). "The prosequence of procaricain forms an alpha-helical domain that prevents access to the substrate-binding cleft". Structure. 4 (10): 1193–203. doi:10.1016/s0969-2126(96)00127-x. PMID8939744.
^Olonen A, Kalkkinen N, Paulin L (July 2003). "A new type of cysteine proteinase inhibitor--the salarin gene from Atlantic salmon (Salmo salar L.) and Arctic charr (Salvelinus alpinus)". Biochimie. 85 (7): 677–81. doi:10.1016/S0300-9084(03)00128-7. PMID14505823.
^Green TB, Ganesh O, Perry K, Smith L, Phylip LH, Logan TM, Hagen SJ, Dunn BM, Edison AS (April 2004). "IA3, an aspartic proteinase inhibitor from Saccharomyces cerevisiae, is intrinsically unstructured in solution". Biochemistry. 43 (14): 4071–81. doi:10.1021/bi034823n. PMID15065849.
^Murai H, Hara S, Ikenaka T, Oda K, Murao S (January 1985). "Amino acid sequence of Streptomyces metallo-proteinase inhibitor from Streptomyces nigrescens TK-23". J. Biochem. 97 (1): 173–80. PMID3888972.
^Ohno A, Tate S, Seeram SS, Hiraga K, Swindells MB, Oda K, Kainosho M (September 1998). "NMR structure of the Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23: another example of an ancestral beta gamma-crystallin precursor structure". J. Mol. Biol. 282 (2): 421–33. doi:10.1006/jmbi.1998.2022. PMID9735297.
^Monteiro AC, Abrahamson M, Lima AP, Vannier-Santos MA, Scharfstein J (November 2001). "Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor in Trypanosoma cruzi". J. Cell Sci. 114 (Pt 21): 3933–42. PMID11719560.
^Figueiredo da Silva AA; de Carvalho Vieira L; Krieger MA; Goldenberg S; Zanchin NI; Guimarães BG (February 2007). "Crystal structure of chagasin, the endogenous cysteine-protease inhibitor from Trypanosoma cruzi". J. Struct. Biol. 157 (2): 416–23. doi:10.1016/j.jsb.2006.07.017. PMID17011790.
^Wang SX, Pandey KC, Scharfstein J, Whisstock J, Huang RK, Jacobelli J, Fletterick RJ, Rosenthal PJ, Abrahamson M, Brinen LS, Rossi A, Sali A, McKerrow JH (May 2007). "The structure of chagasin in complex with a cysteine protease clarifies the binding mode and evolution of an inhibitor family". Structure. 15 (5): 535–43. doi:10.1016/j.str.2007.03.012. PMID17502099.
^Brzin J, Rogelj B, Popovic T, Strukelj B, Ritonja A (June 2000). "Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom clitocybe nebularis". J. Biol. Chem. 275 (26): 20104–9. doi:10.1074/jbc.M001392200. PMID10748021.
^Iwanaga S, Okada M, Isawa H, Morita A, Yuda M, Chinzei Y (May 2003). "Identification and characterization of novel salivary thrombin inhibitors from the ixodidae tick, Haemaphysalis longicornis". Eur. J. Biochem. 270 (9): 1926–34. doi:10.1046/j.1432-1033.2003.03560.x. PMID12709051.